SHAPE and DMS probing of SLNCR1 lncRNA conserved region 403-780

The goal of this study was to understand the underlying structure of nucleotides 403-780 of the lncRNA SLNCR1, in-cell and when extracted from nuclear and cytoplasmic fractions. SHAPE and DMS probing revealed that the region is largely unstructured inside and outside of the cell, and appears protein-bound in primary melanoma cells. RNA from WM1976 (melanoma short-term culture) cells were treated with the SHAPE reagent 5-nitroisatoic anhydride (25 mM) or DMS (2%) either in-cell or after extraction from the cytoplasm or nuclei and removal of proteins. Purified RNA was used as templates in modified reverse transcription conditions (MaP RT), wherein sites of chemical adducts on the RNA were encoded into cDNA as non-templated sequences. MaP RT was initiated with primers specific to SLNCR1 region 403-780 or, as a control, the U1 small nuclear RNA. cDNAs were further amplified, sequenced, and aligned to respective targets. Nucleotide mutation rates were transformed into SHAPE reactivities, which informed RNA structure modeling.